We propose to continue a long-term investigation whose objective is to increase our understanding of the molecular basis of sequence specific protein-DNA recognition mechanisms. Detailed physical-chemical and biological studies of site specific (restriction) endonucleases and plasmid relaxation complexes are proposed which will attempt to define the structural and mechanistic principles underlying protein-DNA recognition interactions in general, as well as the regulation of gene expression. Our research program will involve: (l) the selection of recombinant DNA molecules containing the cloned structural genes of restriction-modification enzymes, the physical mapping and nucleotide sequence analysis of these genes, and the isolation and characterization of mutants; (2) the physical-chemical analysis of restriction endonucleases as proteins, including attempts to grow crystals of at least one restriction enzyme bound to its specific recognition sequence for analysis by X-ray diffraction techniques; (3) the kinetic characterization of DNA hydrolysis with the goal of understanding the mechanism of site-specific cleavage; (4) characterization of the enzyme binding reaction in the absence of hydrolysis, and evaluation of the kinetic and thermodynamic parameters which describe the molecular interactions involved in both specific and non-specific binding. We have developed agarose gel electrophoresis as an analytical method for characterizing DNA structure. We propose to continue and expand our present studies of the quantitative effects of voltage gradient, gel concentration, and ionic conditions on DNA electrophoretic mobility (u) and retardation coefficient (KR). We will concentrate in particular on developing analytical approaches for studying the mechanism of site-specific and conformation-specific DNA binding proteins using this technique.